Highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 RNA levels to below the limit of detection of current assays in many patients, allowing a significant degree of immune reconstitution. However, initial hopes that HAART might permit virus eradication have been dampened by two findings. First, HIV-1 persists in a latent form in a small reservoir of resting memory CD4+ T cells that persist in adults and children. Second, there is evidence for ongoing virus production even in patients who have suppression of viremia to undetectable levels on HAART. The nature of this ongoing virus production and its relationship to the latent reservoir and other viral reservoirs remain unclear. Previous studies from this lab have identified and characterized the latent reservoir for HIV-1 in resting memory CD4+ T cells. The goal of the present project is to understand the nature of ongoing viral production in patients with "undetectable" viral loads on HAART. In particular, we would like to determine the clinical significance of the low level of viremia that is present in patients on HAART who have "undetectable" levels of plasma HIV-1 RNA (<50 copies/ml). A major question is whether the ongoing virus production actually represents new cycles of infection of susceptible cells, with the attendant risk that drug resistance will evolve. In preliminary studies, we have cloned and characterized the RT and protease gene from the low level of plasma viruses present in patients on HAART who have "undetectable" viral loads. These sequences appeared distinctly archival in character. In this proposal, we describe the development of methods for detecting the evolution of drug resistant viruses in patients who have "undetectable levels" of plasma virus. These methods will be used to determine whether the low level of viremia present in patients on HAART with <50 copies/ml of HIV-1 RNA is derived from newly infected cells or from an archival source such as the latent reservoir in resting CD4+ T cells, to test the hypothesis that under optimal conditions current HAART regimens can arrest virus evolution, and to determine whether the analysis of viral evolution can be used to stratify patients on HAART into groups that have clinically significant differences in the control of viral replication and long term outcomes.